Modified Fc molecules

• US 20060140934A1
• Filed: 09/23/2005
• Published: 06/29/2006
• Est. Priority Date: 09/24/2004
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Xiong, Fei, Sitney, Karen C., Gegg, Colin
• Granted Patent: US 7,442,778 B2
• Time in Patent Office: Days
• US Class Current: 424/133.100
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/06 Anabolic agents androgens A61P5/26
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2318/10 Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The present invention concerns molecules and a process in which one or more biologically active peptides are incorporated into an Fc domain. In this invention, pharmacologically active compounds may be prepared by a process comprising (a) selecting at least one peptide that modulates the activity of a protein of interest; and (b) preparing a pharmacologic agent comprising an amino acid sequence of the selected peptide in a loop region of an Fc domain. This process may be employed to modify an Fc domain that is already linked through an N— or C-terminus or sidechain to a peptide or to a polypeptide (e.g., etanercept). This process may also be employed to modify an Fc domain that is part of an antibody (e.g., adalimumab, epratuzumab, infliximab, Herceptin®, and the like). In this way, different molecules can be produced that have additional functionalities, such as a binding domain to a different epitope or an additional binding domain to the precursor molecule'"'"'s existing epitope. The peptide can be selected, for example, by phage display, E. coli display, ribosome display, RNA-peptide screening, yeast-based screening, chemical-peptide screening, rational design, or protein structural analysis.

Claims

1. A composition of matter of the formula

(X¹)_a-F¹-(X²)_b

and multimers thereof, wherein:

F¹ is an Fc domain modified so that it comprises at least one X³ in a loop region;

X¹ and X² are each independently selected from -(L¹)_c-P¹, -(L¹)_c-P¹-(L²)_d-P², -(L¹)_c-P¹-(L²)_d-P²-(L³)_e-P³, and -(L¹)_c-P¹-(L²)_d-P²-(L³)_e-P³-(L⁴)_f-P⁴;

X³ is independently selected from -(L⁵)_c-P⁵, -(L⁵)_c-P⁵-(L⁶)_d-P⁶, -(L⁵)_c-P⁵-(L⁶)_d-P⁶-(L⁷)_e-P⁷, and -(L⁵)_c-P⁵-(L⁶)_d-P⁶-(L⁷)_e-P⁷-(L⁸)_f-P⁸;

P¹, P², P³, and P⁴ are each independently sequences of pharmacologically active polypeptides or pharmacologically active peptides;

P⁵, P⁶, P⁷, and P⁸ are each independently sequences of pharmacologically active peptides;

L¹, L², L³, L⁴, L⁵, L⁶, L⁷, and L⁸ are each independently linkers; and

a, b, c, d, e, and f are each independently 0 or 1.

2. The composition of matter of claim 1 wherein a and b are each 0.

3. The composition of matter of claim 1 wherein the Fc domain comprises an IgG Fc domain.

4. The composition of matter of claim 3 wherein the Fc domain comprises a sequence selected from SEQ ID NOS:

599 and 603 to 607.

5. The composition of matter of claim 1 wherein the Fc domain comprises an IgG1 Fc domain.

6. The composition of matter of claim 5 wherein the IgG1 Fc domain comprises SEQ ID NO:

599 and X³ is inserted into or replaces all or part of a sequence selected from SEQ ID NOS;

621, 622, 624, 625, 627, 628, 630, 632, 634, and 636.

7. The composition of matter of claim 6 wherein X³ is inserted into or replaces all or part of a sequence selected from SEQ ID NOS:

623, 626, 629, 631, 633, 635, and 637.

8. The composition of matter of claim 7 wherein X³ is inserted at Leu₁₃₉/Thr₁₄₀.

9. The composition of matter of claim 5 wherein the IgG1 Fc domain comprises SEQ ID NO:

603 and X³ is inserted into or replaces all or part of a sequence selected from SEQ ID NOS;

621, 622, 624, 625, 627, 628, 630, 632, 634, and 636.

10. The composition of matter of claim 9 wherein X³ is inserted at H₅₃/E₅₄, Y₈₁/N₈₂, N₁₁₀/K₁₁₁, L₁₄₃/T₁₄₄, Q₁₇₁/P₁₇₂, E₁₇₃/N₁₇₄, S₁₈₆/D₁₈₇, G₁₈₈/S₁₈₉, or G₂₀₅/N₂₀₆.

11. The composition of matter of claim 5 wherein the IgG1 Fc domain comprises SEQ ID NO:

604 and X³ is inserted into or replaces all or part of a sequence selected from SEQ ID NOS;

621, 622, 624, 625, 627, 628, 632, 634, 636, and 644.

12. The composition of matter of claim 11 wherein X³ is inserted at H₅₃/E₅₄, Y₈₁/N₈₂, N₁₁₀/K₁₁₁, L₁₄₃/T₁₄₄, Q₁₇₁/P₁₇₂, E₁₇₃/N₁₇₄, S₁₈₆/D₁₈₇, G₁₈₈/S₁₈₉, or G₂₀₅/N₂₀₆.

13. The composition of matter of claim 1 wherein the Fc domain comprises an IgG3 Fc domain.

14. The composition of matter of claim 13 wherein the IgG3 Fc domain comprises SEQ ID NO:

605 and X³ is inserted into or replaces all or part of a sequence selected from SEQ ID NOS;

614, 621, 622, 624, 627, 639, 641, 644, 645, and 646.

15. The composition of matter of claim 14 wherein X³ is inserted at H₁₀₀/E₁₀₁, F₁₂₈/N₁₂₉, N₁₅₇/K₁₅₈, M₁₉₀/T₁₉₁, Q₂₁₈/P₂₁₉, E₂₂₀/N₂₂₁, S₂₃₂/D₂₃₃, G₂₃₄/S₂₃₅, or G₂₅₂/N₂₅₃.

16. The composition of matter of claim 1 wherein the Fc domain comprises an IgG2 Fc domain.

17. The composition of matter of claim 16 wherein the Fc domain comprises SEQ ID NO:

606 and X³ is inserted into or replaces all or part of a sequence selected from SEQ ID NOS;

621, 622, 624, 632, 636, 639, 640, 642, 644, and 646.

18. The composition of matter of claim 17 wherein X³ is inserted at H₄₉/E₅₀, F₇₇/N₇₈, N₁₀₆/K₁₀₇, M₁₃₉/T₁₄₀, Q₁₆₇/P₁₆₈, E₁₆₉/N₁₇₀, S₁₈₁/D₁₈₂, G₁₈₃/S₁₈₄, or G₂₀₁/N₂₀₂.

19. The composition of matter of claim 1 wherein the Fc domain comprises an IgG4 Fc domain.

20. The composition of matter of claim 19 wherein the Fc domain comprises SEQ ID NO:

607 and X³ is inserted into or replaces all or part of a sequence selected from SEQ ID NOS;

620, 621, 624, 627, 632, 634, 638, 639, 643, and 644.

21. The composition of matter of claim 20 wherein X³ is inserted at Q₅₀/E₅₁, F₇₈/N₇₉, N₁₀₇/K₁₀₈, M₁₄₀/T₁₄₁, Q₁₆₈/P₁₆₉, E₁₇₀/N₁₇₁, S₁₈₂/D₁₈₃, G₁₈₄/S₁₈₅, or G₂₀₂/N₂₀₃.

22. The composition of matter of claim 1 wherein the Fc domain comprises SEQ ID NO:

608 and X³ is inserted into or replaces all or part of a sequence selected from SEQ ID NOS;

621, 622, 628, 624, 627, 632, 636, 639, 644, and 646.

23. The composition of matter of claim 22 wherein X³ is inserted at H₁₁₂/E₁₁₃, F₁₄₀/N₁₄₁, N₁₆₉/K₁₇₀, M₂₀₄/T₂₀₅, Q₂₃₂/P₂₃₃, E₂₃₄/N₂₃₅, S₂₄₆/D₂₄₇, G₂₄₈/S₂₄₉, or G₂₆₈/N₂₆₉.

24. The composition of matter of claim 1 wherein X³ comprises an angiotensin-2 (ang-2) binding peptide sequence.

25. The composition of matter of claim 24 wherein the ang-2 binding peptide sequence is selected from SEQ ID NOS:

100 to 189.

26. The composition of matter of claim 25 wherein the ang-2 binding peptide sequence is SEQ ID NO:

147.

27. The composition of matter of claim 26 wherein F¹ comprises an IgG1 Fc domain.

28. The composition of matter of claim 27 having the sequence of SEQ ID NO:

618.

29. The composition of matter of claim 1 wherein X³ comprises a myostatin binding peptide sequence.

30. The composition of matter of claim 29 wherein the myostatin binding peptide sequence is selected from SEQ ID NOS:

218 to 509.

31. The composition of matter of claim 30 wherein the myostatin binding peptide sequence is SEQ ID NO:

365.

32. The composition of matter of claim 31 wherein F¹ comprises an IgG1 Fc domain.

33. The composition of matter of claim 32 having the sequence SEQ ID NO:

612.

34. The composition of matter of claim 1 wherein X³ comprises an erythropoietin-mimetic (EPO-mimetic) peptide sequence.

35. The composition of matter of claim 34 wherein the EPO-mimetic peptide sequence is selected from SEQ ID NOS:

1 to 27.

36. The composition of matter of claim 35 wherein the EPO-mimetic peptide sequence is SEQ ID NO:

2.

37. The composition of matter of claim 36 wherein F¹ comprises an IgG1 Fc domain

38. The composition of matter of claim 37 having the sequence of SEQ ID NO:

615.

39. The composition of matter of claim 1 wherein X³ comprises a thrombopoietin-mimetic (TPO-mimetic) peptide sequence.

40. The composition of matter of claim 39 wherein the TPO-mimetic peptide sequence is selected from SEQ ID NOS:

28 to 99.

41. The composition of matter of claim 40 wherein the TPO-mimetic peptide sequence is SEQ ID NO:

28.

42. The composition of matter of claim 41 wherein F¹ comprises an IgG1 Fc domain

43. The composition of matter of claim 42 having the sequence SEQ ID NO:

616.

44. The composition of matter of claim 1 wherein X³ comprises a nerve growth factor (NGF) binding peptide sequence.

45. The composition of matter of claim 44 wherein the NGF binding peptide sequence is selected from SEQ ID NOS:

190 to 218.

46. The composition of matter of claim 1 wherein X³ comprises a B cell activating factor (BAFF) binding peptide sequence.

47. The composition of matter of claim 46 wherein the BAFF binding peptide sequence is selected from SEQ ID NOS:

510 to 594.

48. A DNA encoding a composition of matter of claim 1.

49. An expression vector comprising the DNA of claim 46.

50. A host cell comprising the expression vector of claim 47.

51. The cell of claim 48, wherein the cell is an E. coli cell.

53. The process of claim 50, wherein the internal region of the Fc domain is a loop region.

54. The process of claim 50, wherein the peptide is selected in a process comprising one or more techniques selected from yeast-based screening, rational design, protein structural analysis, or screening of a phage display library, an E. coli display library, a ribosomal library, or a chemical peptide library.

55. The process of claim 50, wherein the preparation of the pharmacologic agent is carried out by:

a. preparing a gene construct comprising a nucleic acid sequence encoding an Fc domain wherein a sequence the selected peptide is inserted into or replaces one or more amino acids within the Fc domain; and

b. expressing the gene construct.

56. The process of claim 53, wherein the gene construct is expressed in an E. coli cell.

57. The process of claim 50, wherein the protein of interest is a cell surface receptor.

58. The process of claim 50, wherein the protein of interest has a linear epitope.

59. The process of claim 50, wherein the protein of interest is a cytokine receptor.

60. The process of claim 50 wherein the Fc domain is an IgG Fc domain.

52. A process for preparing a pharmacologically active compound, which comprises:

a. selecting at least one randomized peptide that modulates the activity of a protein of interest; and

b. preparing a pharmacologic agent comprising an amino acid sequence of the selected peptide as an internal sequence of an Fc domain.

61. A modified antibody, comprising an Fc domain modified so that it comprises at least one X³ in a loop region, wherein:

X³ is independently selected from -(L⁵)_c-P⁵, -(L⁵)_c-P⁵-(L⁶)_d-P⁶, -(L⁵)_c-P⁵-(L⁶)_d-P⁶-(L⁷)_e-P⁷, and -(L⁵)_c-P⁵-(L⁶)_d-P⁶-(L⁷)_e-P⁷-(L⁸)_f-P⁸;

P⁵, P⁶, P⁷, and P⁸ are each independently sequences of pharmacologically active peptides;

L⁵, L⁶, L⁷, and L⁸ are each independently linkers; and

c, d, e, and f are each independently 0 or 1.

62. A process for preparing a modified antibody, which comprises:

a) selecting at least one peptide that modulates the activity of a protein of interest; and

b) preparing an antibody comprising an amino acid sequence of the selected peptide in a loop region of an Fc domain of the antibody.